NEMO is a protein that is essential for normal innate and adaptive immunity, in addition to homeostasis and the regulation of the pro-inflammatory response. If NEMO is defective, NF-kB transcription factor family members do not translocate to the nucleus to regulate gene transcription, and normal development of the organism, including the immune system, does not occur. In addition to these developmental defects, the immune system does not function correctly if NEMO is defective. This year, we investigated individuals with specific mutations in NEMO and related proteins such as the inhibitor of NF-kappaB, IkappaBalpha, TANK, A20, IKK-beta and U4atac. In addition to clinical and laboratory evaluation of human patients, we obtained patient-derived peripheral blood cells skin fibroblasts to study gene expression and post-translational modifications, biochemically. A reconstitution system utilizing NEMO, A20, and TANK-deficient Jurkat T cells and THP-1 monocytes has also been generated in order to study protein interactions biochemically. By studying cells from our patients, we found that U4atac is important in T cell metabolism, proliferation and activation, because it regulates gene splicing events important to all of these pathways. We characterized the affected pathways in T cells using gene expression and immune cell phenotyping under various differentiation conditions. This work has been prepared for submission to a peer-reviewed journal. We identified a second patient with TANK mutation who also exhibits an immunodeficiency with inflammatory bowel disease phenotype. We determined that TANK mutations associated with disease lead to altered negative regulation of NEMO and NF-kB in monocyte-derived cell lines and T cells. No TANK mutation in humans have been previously described and so this work, which is being prepared for submission to a peer-reviewed journal, will provide novel insights into the regulation of NF-kB and its role in hepatic homeostasis. We identified two additional patients with NEMO-Delta-exon5 mutation who exhibit inflammatory eye disease, skin disease and a type I IFN signature in peripheral blood. Further characterization of the cellular and disease phenotype in these 3 individuals has been completed in response to reviewers comments, which will be submitted to a peer-reviewed journal. We identified two unrelated patients with mutation in IKKbeta that lead to normal levels of protein expression. These individuals are different to the previously described four individuals with IKKbeta in that rather than suffering from primary immunodeficiency, autoimmunity and inflammatory disease are the principal disease phenotypes seen. In similar work, we identified 4 unrelated individuals with IkappaBalpha mutation and inflammatory disease whose mutations would appear to function in a way that defies NF-kappaB signaling dogma namely that IkBa mutations associated with disease lead to impaired IkBalpha degradation and hence impaired NF-kB activation. Ongoing work in this area is targeting previously unappreciated subcellular functions of IkappaBalpha in regulating NF-kB respone genes. Lastly, we have completed a detailed biochemical analysis of NEMO/A20 cross-regulation and how this impacts NF-kB-mediated proinflammatory cytokine production. In this study, we mapped novel ubiquitination sites on both NEMO and A20, characterizing the polyubiquitin forms of the attached chains. We determined that NEMO stabilization occurs in the absence of A20 in response to TNF stimulation, and by a mass spectrometry screen, we identified over 20 E3 ligases recruited by A20 to assist in mediating degradative NEMO polyubiquitination. Insights gained into the role of these mutant proteins and signaling pathways gained from study of these patients with rare diseases will further our understanding of immune system function and regulation.